Field of the Invention
The present invention relates to a method for efficiently recovering nucleic acids having a relatively short chain length such as circulating DNA, from a whole blood sample containing a large amount of long-chain nucleic acids such as genomic DNA.
Description of Related Art
Currently, free nucleic acids circulating in whole blood (peripheral blood) have attracted much attention in the cancer diagnostic field. Circulating nucleic acids (DNA/RNA) in whole blood are used for various purposes, such as for diagnosing carcinomas and residual lesions associated with carcinomas, for assessing the prognosis of carcinomas, for monitoring the disease course of individuals affected by carcinomas, or for observing the effects of anti-cancer therapies on malignant tumors.
It has been reported that circulating nucleic acids (DNA/RNA) in whole blood become damaged, resulting in fragmentation thereof when the circulating nucleic acids are released into peripheral blood from the nucleus, and consequently circulate as a mono- or poly-nucleosomes (structure in which DNA is wound around a nucleosome: histone octamer) in peripheral blood. The length of circulating DNA in whole blood has been reported to vary and has been generally reported to be approximately 100 to 800 bp.
In particular, DNA in a mononucleosome has a very short chain of 148 base pairs, and is short to an extent not comparable with the chain length of blood cell-derived genomic DNA in whole blood. It has been reported to be difficult to recover mononucleosomal DNA due to such a short chain length thereof, when compared with long-chain nucleic acids.
Most of the conventional art methods for purifying nucleic acids are based on one of the following two principles. Many of the methods which have been used from long ago are methods based on a one-step method where a sample containing nucleic acids, such as a whole blood sample, is mixed with a buffer containing a chaotropic agent and an organic extractant to thereby extract nucleic acids. At least one of phenol and chloroform is mainly used as the organic extractant. Impurities such as proteins are discarded together with the organic phase, and the nucleic acids retained in the aqueous phase are isolated and recovered by a phase separation process. This method suffers from problems in that toxic and harmful organic extractants must be used and in that complicated processes are required for the purification of nucleic acids from the aqueous phase recovered by the phase separation process.
In view of the disadvantages of a one-step method, an alternative method to the one-step method has also been established. The alternative method is a method based on selective adsorption of nucleic acids onto a solid carrier (nucleic acid adsorbent) such as silicon dioxide. A sample containing nucleic acids is dissolved if necessary and then brought into contact with a nucleic acid adsorbent to thereby adsorb nucleic acids in the sample onto the nucleic acid adsorbent. The nucleic acids adsorbed onto the nucleic acid adsorbent are eluted by a suitable buffer from the nucleic acid adsorbent. A typical example of such a method may be a Boom method (see, for example, Patent Document 1 (U.S. Pat. No. 5,234,809).). The Boom method is a method in which a sample containing nucleic acids is incubated with a chaotropic buffer and a DNA-binding solid phase (nucleic acid adsorbent) to thereby isolate nucleic acids from the sample. Both extraction of nucleic acids from cells in the sample and adsorption of the extracted nucleic acids onto a nucleic acid adsorbent are realized by a chaotropic buffer. As a method similar to the above-described method, for example, Patent Document 2 (PCT International Publication No. WO 1993/11221) also discloses a method in which a sample containing nucleic acids is brought into contact with an anion exchanger in a low ionic strength buffer to adsorb nucleic acids in the sample, the nucleic acids eluted from the anion exchanger using a high ionic strength buffer are adsorbed again onto an inorganic carrier member in the presence of a lower alcohol, and then the nucleic acids are recovered by water or a low ionic strength buffer from the inorganic carrier member.
Nucleic acids having a relatively longer chain length than nucleic acids having a short chain length are readily adsorbed onto a nucleic acid adsorbent. Therefore, in the case where nucleic acids are recovered from a whole blood sample using such a method employing a nucleic acid adsorbent, blood cell-derived long chain DNA will be more preferentially recovered than circulating DNA having a short chain length, whereby it is difficult to selectively recover circulating DNA. For this reason, circulating DNA has been conventionally recovered by carrying out a nucleic acid recovery method utilizing a nucleic acid adsorbent for plasma or serum where blood cell components had been previously removed from a whole blood sample. However, there are problems that in this case a special apparatus such as a centrifuge is necessary to recover plasma or serum and the recovery method cannot be carried out if not under a well-equipped environment.
Regarding a technique for concentrating short-chain nucleic acids or a technique for separating short-chain nucleic acids from long-chain nucleic acids, for example, Patent Document 3 (Published Japanese Translation No. 2011-522529 of the PCT International Publication) discloses a method in which a sample containing nucleic acids is brought into contact with a nucleic acid adsorbent in the presence of at least one chaotropic compound and 25 to 35 volume % of at least one of a branched alcohol and an unbranched alcohol, and then the nucleic acids adsorbed onto the nucleic acid adsorbent are eluted. Further, Patent Document 4 (PCT International Publication No. WO 2007/065934) discloses a method in which short-chain nucleic acids are preferentially adsorbed onto a nucleic acid adsorbent by a combined use of citrate rather than a chaotropic salt, and an alcohol.
An object of the present invention is to provide a method of efficiently recovering short-chain DNA having a length of 1000 bases or less, such as circulating DNA, directly from a whole blood sample without requiring serum separation or plasma separation, and a kit used in the same method.